Project Summary/Abstract The proteome reflects the physiology and pathology states of a patient therefore proteomics is a powerful tool for early diagnostics of diseases and monitoring of therapeutic responses. Mass spectrometry (MS) measures the mass-to-charge ratio of charged species and has become the enabling technology for proteomics. However, the majority of the current proteomics studies rely on bottom-up/shotgun approaches. In this case, mixtures of proteins are digested by one of the proteases (e.g., trypsin), separated by liquid chromatography (LC), and analyzed by electrospray mass spectrometry (ESI-MS). Despite tremendous successes, there remain two major limitations in bottom-up proteomics: first, it is difficult to identify all protein isoforms or proteoforms, including splicing, modifications, cleavages, etc.; second, the native state of proteins is always lost after digestion. There is currently a great push to implement top-down proteomics, i.e., identification and characterization of full-length proteins by LC-MS. Unfortunately, top-down proteomics proves to be much more challenging. There are several bottlenecks: first, lower MS sensitivity of protein relative to peptides; second, limitation on detection of high molecular weight proteins; third, inefficient identification of proteins by MS/MS fragmentation; and fourth, laborious multidimensional protein separation not suitable for small volumes of biological samples. The field is calling for transformative technologies. In response to PA-11-215, Newomics Inc. proposes to develop a new technology, picoelectrospray ionization mass spectrometry (picoESI-MS), based on our breakthrough multinozzle emitter array, for top-down proteomics of small-volume samples. The technology will be built on our microfabricated monolithic multinozzle emitters (M3 emitters) and multinozzle emitter array (MEA) chips for LC-nanoESI-MS, which collectively offer a straightforward yet novel solution to the longstanding problem of the efficient coupling between silicon microfluidic chips and ESI-MS, and pave the way for the large-scale integration on the proposed microfluidic chips for LC-picoESI-MS. Our picoESI-MS platform will directly address the aforementioned bottlenecks and thus enable high-sensitivity, high-throughput, and multiplex top-down proteomics of small volumes of biological samples.